Micro-fluidic chip and cell counting system

Abstract

The invention relates to the technical field of cell packaging and counting, in particular to a micro-fluidic chip and a cell counting system. A sample hole, a dye hole, a waste liquid hole and a dilution hole are formed in the micro-fluidic chip, a sample micro-channel, a dye micro-channel, a waste liquid channel and a dilution micro-channel are arranged in the micro-fluidic chip, the sample hole is communicated with the sample micro-channel, the dye hole is communicated with the dye micro-channel, the waste liquid hole is communicated with the waste liquid channel, and the dilution hole is communicated with the dilution micro-channel; a dilution cavity, a uniform mixing micro-channel and a photographing observation cavity are also arranged in the micro-fluidic chip. According to the micro-fluidic chip disclosed by the invention, through integrated design and ingenious design of the positions and connection relations of the dilution cavity, the uniform mixing micro-channel and the photographing observation cavity, the steps of sample dilution, dyeing, observation and the like are successfully integrated into one micro-fluidic chip, so that a full-automatic process from sample introduction to final counting is realized, the operation flow is greatly simplified, and the working efficiency is improved. And the cell counting efficiency and accuracy are greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell detection, in particular to a microfluidic chip and a cell counting system. BACKGROUND

[0002] The microfluidic chip is a system integrating microfluidic channels, reaction chambers and detection units, which can precisely manipulate microfluids on a micron scale (usually tens to hundreds of microns). Its core technology combines microfabrication, fluid mechanics, biochemistry and electronic engineering, and has the characteristics of high throughput, low consumption and fast response, and is widely used in biomedical, chemical analysis, environmental monitoring and other fields.

[0003] In a large number of cell biological research experiments, the concentration of cells or other biological microparticles needs to be detected (quantitative counting), and the concentration or quantity of cell suspension is not only a monitoring parameter of cell culture, but also a necessary parameter in many experimental projects. It is a very basic but important process condition for the successful completion of the experiment.

[0004] The existing cell counting means mainly counts by taking pictures of the microfluidic chip. This counting method usually needs to be diluted, mixed and dyed by manual operation before taking pictures, and needs to rely on manual operation of multiple steps for processing, which has low automation degree and large workload of the operator. SUMMARY

[0005] The purpose of the present application is to provide a microfluidic chip and a cell counting system to improve the automation degree of cell counting and reduce the workload of the operator.

[0006] The present application provides a microfluidic chip, which is provided with a sample hole, a dye hole, a waste liquid hole and a dilution hole, and is provided with a sample microfluidic channel, a dye microfluidic channel, a waste liquid channel and a dilution microfluidic channel inside, the sample hole is in communication with the sample microfluidic channel, the dye hole is in communication with the dye microfluidic channel, the waste liquid hole is in communication with the waste liquid channel, and the dilution hole is in communication with the dilution microfluidic channel.

[0007] The microfluidic chip is further provided with a dilution cavity, a mixing microfluidic channel and a photographing observation cavity inside.

[0008] The dilution microfluidic channel and the sample microfluidic channel are both in communication with the inlet of the dilution cavity, and the outlet of the dilution cavity and the dye microfluidic channel are both in communication with the mixing microfluidic channel, so as to dye the sample in the mixing microfluidic channel.

[0009] The inlet of the photographing observation cavity is in communication with the mixing microfluidic channel, and the outlet of the photographing observation cavity is in communication with the waste liquid channel.

[0010] Further, the microfluidic chip comprises a first cover plate and a second cover plate which are attached to each other, the first cover plate is provided with a first sink, the second cover plate is provided with a second sink, and the first sink and the second sink are oppositely arranged and surround the dilution cavity.

[0011] Further, the dilution cavity is provided with a cell filter layer, the cell filter layer is clamped between the first sink and the second sink, and the pore size of the cell filter layer is set according to the size of the target substance to be filtered in the sample.

[0012] Further, the first sink and the second sink are both provided with a micro-column structure, the micro-column structure comprises a plurality of spaced-apart protrusions, and the cell filter layer is clamped between the micro-column structure of the first sink and the micro-column structure of the second sink.

[0013] Further, the first sink or the second sink is provided with a relief ring groove for avoiding the cell filter layer, and the edge of the cell filter layer is embedded in the relief ring groove.

[0014] Further, the mixing microfluidic channel comprises a plurality of mixing units connected in sequence, and the mixing units are used to guide the liquid to realize splitting and folding.

[0015] Further, the photographing observation cavity is provided with a lens focusing feature and a single cell fluid channel.

[0016] The single cell fluid channel and the lens focusing feature have a preset height difference, the lens focusing feature is used to determine the focal length of the lens, and the size of the single cell fluid channel is set according to the size of the cells to be counted in the sample.

[0017] The application also provides a cell counting system, comprising the microfluidic chip according to any one of the above technical solutions, and further comprising a sample container, a dilution container, a dye container, a waste liquid container, a first pumping element, a second pumping element, a third pumping element, a first pipeline, a second pipeline, a third pipeline and a fourth pipeline, the sample container is communicated with the sample hole through the first pipeline, the first pumping element is arranged on the first pipeline, the dilution container is communicated with the dilution hole through the second pipeline, the second pumping element is arranged on the second pipeline, the dye container is communicated with the dye hole through the third pipeline, the third pumping element is arranged on the third pipeline, and the waste liquid container is communicated with the waste liquid hole through the fourth pipeline.

[0018] Further, a fifth pipeline is provided, a switching valve is arranged at the sample hole, the fifth pipeline is connected with the end of the first pipeline through the switching valve, the switching valve has a first state and a second state, in the first state, the first pipeline is communicated with the fifth pipeline and is not communicated with the sample hole, in the second state, the first pipeline is communicated with the sample hole and is not communicated with the fifth pipeline.

[0019] Further, a one-way valve is arranged on the fourth pipeline and the fifth pipeline, the one-way valve on the fourth pipeline is arranged between the microfluidic chip and the sample container and between the sample container and the first pumping element.

[0020] Compared with the prior art, the present application has at least the following beneficial effects:

[0021] The microfluidic chip of the present application successfully integrates the functions of sample dilution, staining and observation into one microfluidic chip through integrated design, ingenious design of the positions and connection relationships of the dilution cavity, the mixing microfluidic channel and the photographing observation cavity, realizes the fully automatic process from sample introduction to final counting, greatly simplifies the operation process and greatly improves the efficiency and accuracy of cell counting. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a structural schematic diagram of one embodiment of the microfluidic chip in the present application;

[0023] Figure 2 is a parts explosion diagram of the microfluidic chip in the present application; Figure 1

[0024] Figure 3 is a parts explosion diagram of the microfluidic chip in the present application from another perspective; Figure 1

[0025] Figure 4 is a structural schematic diagram of the first cover plate of the microfluidic chip in the present application; Figure 1

[0026] Figure 5 is a structural schematic diagram of the second cover plate of the microfluidic chip in the present application; Figure 1

[0027] Figure 6 is a local enlarged view of A of the second cover plate in the present application and a schematic diagram of multiple embodiments of the mixing unit; Figure 5

[0028] Figure 7 is a structural schematic diagram of one embodiment of the cell counting system of the present application.

[0029] REFERENCE SIGNS: ​​​​​

[0030] 1, sample hole; 2, dye hole; 3, waste hole; 4, dilution hole;

[0031] 5, sample microfluidic channel; 6, dye microfluidic channel; 7, waste fluid channel; 8, dilution microfluidic channel;

[0032] 9, mixing microfluidic channel; 10, photographing observation cavity; 11, cell filter layer; 12, first cover plate; 13, second cover plate; 14, first sink; 15, second sink; 16, lens focusing feature; 17, avoidance ring groove; 18, mixing unit; 181, prismatic; 182, Tesla valve type; 183, concentric circle type;

[0033] 20, microfluidic chip; 21, sample container; 22, dilution container; 23, dye container; 24, waste fluid container; 25, first pumping element; 26, second pumping element; 27, third pumping element; 28, first pipeline; 29, second pipeline; 30, third pipeline; 31, fourth pipeline; 32, first pinch valve; 33, second pinch valve; 34, fifth pipeline; 35, switching valve. DETAILED DESCRIPTION

[0034] A microfluidic chip and a cell counting system according to the present application will be described below with reference to the accompanying drawings, in which the preferred embodiments of the present application are shown, it should be understood that those skilled in the art can modify the present application described herein while still achieving the advantageous effects of the present application. Therefore, the following description should be understood as a broad knowledge for those skilled in the art, and not as a limitation on the present application.

[0035] The serial numbers of components in this paper, such as "first", "second", etc., are only used to distinguish the described objects, and have no technical meaning. The "connection" and "coupling" in this application include direct and indirect connection (coupling) unless otherwise specified. In the description of the present application, it should be understood that the terms "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, therefore it cannot be understood as a limitation on the present application.

[0036] In the present application, unless otherwise explicitly specified and limited, a first feature is "on" or "under" a second feature can be that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature is "over", "above" and "on top of" the second feature can be that the first feature is directly above or obliquely above the second feature, or only means that the first feature is horizontally higher than the second feature. The first feature is "under", "below" and "underneath" the second feature can be that the first feature is directly below or obliquely below the second feature, or only means that the first feature is horizontally lower than the second feature.

[0037] The present application will be described in more detail by way of example with reference to the accompanying drawings. The advantages and features of the present application will become more apparent from the following description and claims. It should be noted that the drawings are in a very simplified form and are not drawn to precise scale, only for the purpose of facilitating, clarifying and assisting in the explanation of the embodiments of the present application.

[0038] The present application will be described in more detail by way of example with reference to the accompanying drawings. The advantages and features of the present application will become more apparent from the following description and claims. It should be noted that the drawings are in a very simplified form and are not drawn to precise scale, only for the purpose of facilitating, clarifying and assisting in the explanation of the embodiments of the present application. Figures 1 to 7 The microfluidic chip and cell counting system of the present application are introduced.

[0039] As shown in Figure 1 , Figure 2 and Figure 3 , the present application provides a microfluidic chip, which is provided with a sample hole 1, a dye hole 2, a waste liquid hole 3 and a dilution hole 4, and is provided with a sample microfluidic channel 5, a dye microfluidic channel 6, a waste liquid channel 7 and a dilution microfluidic channel 8 inside the microfluidic chip, the sample hole 1 is in communication with the sample microfluidic channel 5, the dye hole 2 is in communication with the dye microfluidic channel 6, the waste liquid hole 3 is in communication with the waste liquid channel 7, and the dilution hole 4 is in communication with the dilution microfluidic channel 8; the microfluidic chip is further provided with a dilution cavity, a mixing microfluidic channel 9 and a photographing observation cavity 10; the dilution microfluidic channel 8 and the sample microfluidic channel 5 are both in communication with the inlet of the dilution cavity, the outlet of the dilution cavity and the dye microfluidic channel 6 are both in communication with the mixing microfluidic channel 9, so as to dye the sample in the mixing microfluidic channel 9; the inlet of the photographing observation cavity 10 is in communication with the mixing microfluidic channel 9, and the outlet of the photographing observation cavity 10 is in communication with the waste liquid channel 7.

[0040] Among them, the microfluidic chip refers to a device for manipulating and analyzing microfluids on a micrometer scale, which can be made by microfabrication techniques such as photolithography and soft lithography.

[0041] The sample hole 1 refers to an opening for introducing a sample to be analyzed, which can be designed in a circular or square shape, and the diameter or side length is usually in the range of 1-5 millimeters.

[0042] Dye well 2 refers to the opening for introducing dye, which can be designed similarly to sample well 1. The dye can be AOPI dye (Acridine Orange / Propidium Iodide), a fluorescent dye specially used for detecting and measuring DNA content.

[0043] Waste well 3 refers to the opening for discharging the processed liquid, which can be designed with a larger diameter to ensure smooth drainage.

[0044] Dilution well 4 refers to the opening for introducing dilution liquid, which can also be designed similarly to sample well 1.

[0045] Microchannel refers to the micro channel inside the chip for liquid flow, which can be designed with rectangular or circular cross section, usually with a width in the range of 10-500 microns.

[0046] Dilution chamber refers to the space for mixing sample and dilution liquid, which can be designed with an expanded chamber, with micro-column structures added inside to enhance mixing effect.

[0047] Mixing microchannel 9 refers to the channel for mixing the diluted sample and dye, which can be designed with a serpentine or zigzag shape to increase the degree of fluid mixing.

[0048] Photographing observation chamber 10 refers to the observation area for cell counting, which can be designed with a flattened design to ensure that the cells are distributed on the same focal plane.

[0049] Among them, sample well 1 connects sample microchannel 5, which is used to introduce the cell sample to be analyzed; dye well 2 connects dye microchannel 6, which is used to introduce the staining reagent; waste well 3 connects waste flow channel 7, which is used to discharge the processed waste liquid; dilution well 4 connects dilution microchannel 8, which is used to introduce dilution liquid.

[0050] The chip is internally provided with three key functional areas: dilution chamber, mixing microchannel 9 and photographing observation chamber 10. The dilution chamber is connected with dilution microchannel 8 and sample microchannel 5, which is used to mix the sample and dilution liquid. Mixing microchannel 9 connects dilution chamber and dye microchannel 6, which is used to mix the diluted sample with dye. Photographing observation chamber 10 connects mixing microchannel 9 and waste flow channel 7, which is used for final cell observation and counting.

[0051] In operation, first, the sample and the diluent are introduced into the dilution chamber through the sample hole 1 and the diluent hole 4 respectively. In the dilution chamber, the sample is sufficiently diluted. Then, the diluted sample flows into the mixing micro-channel 9, while the dye is introduced through the dye hole 2. In the mixing micro-channel 9, the diluted sample and the dye are mixed sufficiently. Finally, the dyed sample enters the photographing observation chamber 10, where the cell counting is performed. After the counting is completed, the sample is discharged through the waste liquid channel 7. It should be noted that if the sample does not need to be diluted, the diluent does not need to be introduced into the dilution chamber.

[0052] By such an integrated design, the positions and connection relationships of the dilution chamber, the mixing micro-channel 9 and the photographing observation chamber 10 are ingeniously designed, the steps of sample dilution, dyeing and observation are successfully integrated into one micro-fluidic chip, the fully automatic process from sample introduction to final counting is realized, the operation process is greatly simplified, and the efficiency and accuracy of cell counting are greatly improved.

[0053] In some embodiments, as shown in Figs. 1-3, the micro-fluidic chip for cell counting comprises a first cover plate 12 and a second cover plate 13 which are attached to each other, and the dilution chamber is formed by the first and second cover plates 12 and 13. Figure 3 Figure 4 Figure 5 In some embodiments, as shown in Figs. 1-3, the micro-fluidic chip for cell counting comprises a first cover plate 12 and a second cover plate 13 which are attached to each other, and the dilution chamber is formed by the first and second cover plates 12 and 13.

[0054] In some embodiments, the first cover plate 12 and the second cover plate 13 are made of a material with a light transmittance of more than 90%, such as polydimethylsiloxane (PDMS) material, and the first cover plate 12 and the second cover plate 13 can be combined by thermal bonding or ultrasonic bonding, etc., with a bonding strength of more than 0.5 MPa.

[0055] In some embodiments, a cell filter layer 11 for filtering the sample is arranged in the dilution chamber, the cell filter layer 11 is clamped between the first and second recesses 14 and 15, and the pore size of the cell filter layer 11 is set according to the size of the target substance to be filtered in the sample.

[0056] By arranging the cell filter layer 11 in the dilution chamber, the sample can be filtered before entering the mixing micro-channel 9 for dyeing. This design integrates the filtering step into the micro-fluidic chip, eliminating the need for additional external filtering equipment, simplifying the operation process and improving the degree of automation. At the same time, the filtered sample can directly enter the subsequent dyeing and observation steps, reducing the possibility of contamination or loss during sample transfer.

[0057] ​​Specifically, a porous membrane can be used as the filter layer, and the pore size can be selected according to the target substance to be filtered. In addition, the material selection of the cell filter layer 11 is also important. A material with good biocompatibility, such as polycarbonate, polyether sulfone, etc., can be selected to ensure that no contamination or influence is caused to the sample. Further, in some embodiments, a micro-pillar structure is arranged in the first sink 14 and the second sink 15, the micro-pillar structure includes a plurality of spaced-apart protrusions, and the cell filter layer 11 is clamped between the micro-pillar structures of the first sink 14 and the second sink 15.

[0058] The design of the micro-pillar structure not only provides stable support for the cell filter layer 11, but also does not affect the filtering function, and can also disperse the sample liquid, which is beneficial to the uniform dispersion and dilution of the sample liquid. This structure design is simple and ingenious, easy to process and assemble, and improves the performance and reliability of the microfluidic chip. The micro-pillar structure can be processed by etching.

[0059] In some embodiments, an avoidance ring groove 17 for avoiding the cell filter layer 11 is arranged around the first sink 14 or the second sink 15, and the edge of the cell filter layer 11 is embedded in the avoidance ring groove.

[0060] Specifically, the avoidance ring groove 17 can be arranged around the first sink 14, around the second sink 15, or around both the first sink 14 and the second sink 15. The shape of the avoidance ring groove 17 can be a circular ring, a square ring, or other suitable shapes to match the outer shape of the cell filter layer 11, so that the edge of the cell filter layer 11 is embedded in the avoidance ring groove 17. The depth of the avoidance ring groove 17 can be designed according to the thickness of the cell filter layer 11, and is usually slightly larger than the thickness of the cell filter layer 11, so as to ensure that the cell filter layer 11 can be completely embedded in the avoidance ring groove 17.

[0061] In some embodiments, in order to mix the sample and the dye sufficiently and uniformly, as shown in Figure 5 and Figure 6 , the mixing microfluidic channel 9 includes a plurality of mixing units 18 connected in sequence, and the mixing unit 18 is used to guide the liquid to realize splitting and folding.

[0062] Specifically, as shown in Figure 6 , the mixing unit 18 can be a prismatic shape 181, a Tesla valve type 182, or a concentric circle type 183, to guide the liquid to realize splitting and folding. In order to improve the mixing effect, the mixing microfluidic channel 9 is arranged in the form of a coil.

[0063] Further, the cross sections of the dilution chamber and the photographing observation chamber are both rhombic. In this way, a converging-diverging flow path (i.e., a flow path having both a converging section and a diverging section) can be formed to increase fluid disturbance, thereby improving mixing effect.

[0064] In some embodiments, in order to facilitate determination of the focal length of the lens, a lens focusing feature 16 and a single-cell fluid channel are arranged in the photographing observation chamber 10; the single-cell fluid channel has a preset height difference with the lens focusing feature, the lens focusing feature 16 is used to determine the focal length of the lens, and the size of the single-cell fluid channel is set according to the size of the target cells (i.e., the cells to be counted) in the sample.

[0065] Specifically, the lens focusing feature 16 is arranged on the bottom side wall of the photographing observation chamber 10, and the lens focusing feature 16 can be a cross line, a circle or other geometric patterns; the single-cell fluid channel has a preset height difference with the lens focusing feature 16; the depth of the lens focusing feature 16 can be set to be higher or lower than the depth of the single-cell fluid channel by a distance to form the preset height difference, for example, when the depth of the lens focusing feature 16 is 10 microns higher than that of the single-cell fluid channel, the focusing of the single-cell fluid channel can be completed by moving the photographing observation chamber 10 by 10 microns after focusing on the lens focusing feature 16; when the depth of the lens focusing feature 16 is 10 microns lower than that of the single-cell fluid channel, the focusing of the single-cell fluid channel can be completed by moving the photographing observation chamber 1010 microns away after focusing on the lens focusing feature 16. The diameter of the single-cell fluid channel is set according to the size of the cells to be counted in the sample.

[0066] The arrangement of the lens focusing feature 16 can facilitate automatic focusing, so that the microfluidic chip can better cooperate with other automated equipment to realize full-process automation from sample preparation to data analysis, which not only improves experimental efficiency but also reduces human error and provides the possibility for high-throughput cell analysis.

[0067] The application also provides a cell counting system, which comprises the microfluidic chip. Figure 7As shown, the cell counting system comprises a microfluidic chip 20, which has the same structure as the microfluidic chip in any of the above technical solutions, and will not be described here again. The cell counting system further comprises a sample container 21, a dilution container 22, a dye container 23, a waste liquid container 24, a first pumping element 25, a second pumping element 26, a third pumping element 27, a first pipeline 28, a second pipeline 29, a third pipeline 30, and a fourth pipeline 31. The sample container 21 is in communication with the sample hole through the first pipeline 28, and the first pumping element 25 is arranged on the first pipeline 28. The dilution container 22 is in communication with the dilution hole 4 through the second pipeline 29, and the second pumping element 26 is arranged on the second pipeline 29. The dye container 23 is in communication with the dye hole 2 through the third pipeline 30, and the third pumping element 27 is arranged on the third pipeline 30. The waste liquid container 24 is in communication with the waste liquid hole 3 through the fourth pipeline 31.

[0068] Specifically, in the cell counting system of the present application, the sample container 21 is in communication with the sample hole on the microfluidic chip 20 through the first pipeline 28, and the first pumping element 25 is arranged on the first pipeline 28. This arrangement can achieve automatic delivery and precise control of the sample. The first pumping element 25 can adopt a micro-injection pump or a peristaltic pump, etc., and the appropriate pumping type and flow range are selected according to actual needs. A first pinch valve 32 is further arranged on the first pipeline 28 to control the opening and closing of the first pipeline 28, and the first pinch valve 32 is located upstream of the first pumping element 25 and is a normally closed pinch valve.

[0069] The dilution container 22 is in communication with the dilution hole 4 on the microfluidic chip 20 through the second pipeline 29, and the second pumping element 26 is arranged on the second pipeline 29. This arrangement can achieve automatic delivery and precise control of the dilution liquid. The second pumping element 26 can adopt a micro-injection pump or a peristaltic pump, etc., and the appropriate pumping type and flow range are selected according to actual needs.

[0070] The dye container 23 is in communication with the dye hole 2 on the microfluidic chip 20 through the third pipeline 30, and the third pumping element 27 is arranged on the third pipeline 30. This arrangement can achieve automatic delivery and precise control of the dye. The third pumping element 27 can adopt a micro-injection pump or a peristaltic pump, etc., and the appropriate pumping type and flow range are selected according to actual needs.

[0071] The waste liquid container 24 is in communication with the waste liquid hole 3 on the microfluidic chip 20 through the fourth pipeline 31, which can achieve automatic collection of waste liquid and avoid pollution. A second pinch valve 33 is arranged on the fourth pipeline 31 to control the opening and closing of the fourth pipeline 31, and the second pinch valve 33 is a normally open pinch valve.

[0072] The cooperation of these components can realize the automatic processing of the sample. First, the first pumping member 25 transports the sample from the sample container 21 to the sample hole of the microfluidic chip 20. Then, the second pumping member 26 transports the diluent from the diluent container 22 to the dilution hole 4 of the microfluidic chip 20, mixes with the sample inside the microfluidic chip 20, and realizes the automatic dilution of the sample. Next, the third pumping member 27 transports the dye from the dye container 23 to the dye hole 2 of the microfluidic chip 20, mixes with the diluted sample, realizes the automatic staining, and the stained sample enters the photographing observation cavity 10 and is photographed by the photographing device. Finally, the sample after photographing is discharged into the waste liquid container 24 through the fourth pipeline 31.

[0073] The cell counting system of the present application realizes the automatic transportation, dilution, staining, photographing and waste liquid collection of the sample by setting multiple containers, pumping members and pipelines, greatly improving the automation degree of the cell counting process. Through the precise control of the pumping member, the precise quantification and dilution of the sample can be realized, and the accuracy of the counting is improved. At the same time, the automatic system reduces the manual operation, reduces the experimental error, and improves the experimental efficiency.

[0074] In some embodiments, in order to prevent the residual cell liquid in the first pipeline 28 from affecting the subsequent detection, it is necessary to update the cell liquid in the first pipeline 28, and the cell counting system further comprises a fifth pipeline 34, a switching valve 35 is arranged at the sample hole 1, the fifth pipeline 34 is connected with the end of the first pipeline 28 through the switching valve 35, and the switching valve 35 has a first state and a second state. In the first state, the first pipeline 28 communicates with the fifth pipeline 34 and does not communicate with the sample hole 1, and in the second state, the first pipeline 28 communicates with the sample hole 1 and does not communicate with the fifth pipeline 34. Preferably, the switching valve 35 is a two-way pinch valve.

[0075] In order to prevent the backflow of the liquid, ensure the one-way flow of the system, and improve the stability and reliability of the system, a one-way valve is arranged on the fourth pipeline 31 and the fifth pipeline 34.

[0076] According to different use requirements, the cell counting system of the present application can perform different operations, which are as follows:

[0077] When the sample in the sample container 21 is low concentration cells, no dilution is needed, the third pumping member 27 is not working, the first pinch valve 32 is opened, the first pumping member 25 is working, the two-way pinch valve is in the second state, the first pipeline 28 is in communication with the sample hole 1 and not in communication with the fifth pipeline 34, the cell liquid enters the microfluidic chip 20, and the second pumping member 26 is working, the dye in the dye container 23 enters the microfluidic chip 20, the cell liquid mixes with the dye in the mixing microchannel 9 and then enters the photographing observation cavity 10 for photographing, and then is discharged into the waste liquid container 24 through the fourth pipeline 31.

[0078] When the sample in the sample container 21 is high concentration cells, dilution is needed, the first pinch valve 32 is opened, the first pumping member 25 and the third pumping member 27 are working, the two-way pinch valve is in the second state, the first pipeline 28 is in communication with the sample hole 1 and not in communication with the fifth pipeline 34, the cell liquid and the diluent enter the microfluidic chip 20 and mix, so that the cell liquid is diluted, the second pumping member 26 is working, the dye in the dye container 23 enters the microfluidic chip 20, the cell liquid mixes with the dye in the mixing microchannel 9 and then enters the photographing observation cavity 10 for photographing, and then is discharged into the waste liquid container 24 through the fourth pipeline 31.

[0079] When different samples need to be counted, the cell liquid of the previous batch of samples remaining in the pipeline needs to be discharged, specifically, the first pinch valve 32 and the switching valve 35 are opened, the first pipeline 28 is in communication with the fifth pipeline 34 and not in communication with the sample hole 1, and the first pumping member 25 is working, so that the cell liquid remaining in the first pipeline 28 is discharged into the waste liquid bag by the sample container 21, in this process, the second normally open pinch valve is closed, and the second pumping member 26 is not working.

[0080] When the chip needs to be cleaned, the first normally closed pinch valve, the first pumping member 25, the second normally open pinch valve, the second pumping member 26 and the switching valve 35 are not working, the third pumping member 27 is working, the diluent in the diluent container 22 enters the microfluidic chip 20 and flushes the microfluidic chip 20, and the flushed diluent is discharged into the waste liquid container 24.

[0081] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A microfluidic chip, characterized by, The microfluidic chip is provided with a sample hole, a dye hole, a waste liquid hole and a dilution hole, and is provided with a sample microchannel, a dye microchannel, a waste liquid channel and a dilution microchannel inside, the sample hole is communicated with the sample microchannel, the dye hole is communicated with the dye microchannel, the waste liquid hole is communicated with the waste liquid channel, and the dilution hole is communicated with the dilution microchannel; The microfluidic chip is provided with a sample hole, a dye hole, a waste liquid hole and a dilution hole, and is provided with a sample microchannel, a dye microchannel, a waste liquid channel and a dilution microchannel inside, the sample hole is communicated with the sample microchannel, the dye hole is communicated with the dye microchannel, the waste liquid hole is communicated with the waste liquid channel, and the dilution hole is communicated with the dilution microchannel; The dilution microchannel and the sample microchannel are both communicated with the inlet of the dilution cavity, the outlet of the dilution cavity and the dye microchannel are both communicated with the mixing microchannel, so as to dye the sample in the mixing microchannel; The inlet of the photographing observation cavity is communicated with the mixing microchannel, and the outlet of the photographing observation cavity is communicated with the waste liquid channel.

2. The microfluidic chip of claim 1, wherein, The microfluidic chip comprises a first cover plate and a second cover plate which are attached to each other, the first cover plate is provided with a first sink, the second cover plate is provided with a second sink, and the first sink and the second sink are oppositely arranged and surround the dilution cavity.

3. The microfluidic chip of claim 2, wherein, The dilution cavity is provided with a cell filter layer, the cell filter layer is clamped between the first sink and the second sink, and the pore size of the cell filter layer is set according to the size of the target substance to be filtered in the sample.

4. The microfluidic chip of claim 3, wherein, The first sink and the second sink are both provided with a microcolumn structure, the microcolumn structure comprises a plurality of spaced-apart protrusions, and the cell filter layer is clamped between the microcolumn structure of the first sink and the microcolumn structure of the second sink.

5. The microfluidic chip of claim 3, wherein, The periphery of the first sink or the second sink is provided with a avoiding ring groove for avoiding the cell filter layer, and the edge of the cell filter layer is embedded in the avoiding ring groove.

6. The microfluidic chip of claim 1, wherein, The mixing microchannel comprises a plurality of mixing units connected in sequence, and the mixing units are used for guiding liquid to realize flow separation and folding.

7. The microfluidic chip of claim 1, wherein, The photographing observation cavity is provided with a lens focusing feature and a single cell fluid channel; The single cell fluid channel has a preset height difference with the lens focusing feature, the lens focusing feature is used for determining the focal length of the lens, and the size of the single cell fluid channel is set according to the size of the cells to be counted in the sample.

8. A cell counting system characterized by, The microfluidic chip comprises a sample container, a dilution container, a dye container, a waste liquid container, a first pumping element, a second pumping element, a third pumping element, a first pipeline, a second pipeline, a third pipeline and a fourth pipeline, the sample container is communicated with the sample hole through the first pipeline, the first pumping element is arranged on the first pipeline, the dilution container is communicated with the dilution hole through the second pipeline, the second pumping element is arranged on the second pipeline, the dye container is communicated with the dye hole through the third pipeline, the third pumping element is arranged on the third pipeline, and the waste liquid container is communicated with the waste liquid hole through the fourth pipeline.

9. The cell counting system of claim 8, wherein, A fifth pipeline is further included, a switching valve is arranged at the sample hole, the fifth pipeline is connected with the end of the first pipeline through the switching valve, the switching valve has a first state and a second state, in the first state, the first pipeline is in communication with the fifth pipeline and is not in communication with the sample hole, in the second state, the first pipeline is in communication with the sample hole and is not in communication with the fifth pipeline.

10. The cell counting system of claim 9, wherein, A one-way valve is arranged on each of the fourth pipeline and the fifth pipeline, and a pinch valve is arranged between the one-way valve on the fourth pipeline and the microfluidic chip and between the sample container and the first pumping element.

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